A Distinct Pathway for Polar Exocytosis in Plant Cell
Wall Formation1[OPEN]
Hao Wang 2, Xiaohong Zhuang 2, Xiangfeng Wang, Angus Ho Yin Law, Teng Zhao, Shengwang Du,
Michael M.T. Loy, and Liwen Jiang*
School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of
Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China (H.W.,
X.Z., X.W., A.H.Y.L., L.J.); College of Life Sciences, South China Agricultural University, Guangzhou 510642,
China (H.W.); Department of Physics, The Hong Kong University of Science and Technology, Clear Water Bay,
Kowloon, Hong Kong, China (T.Z., S.D., M.M.T.L.); Division of Biomedical Engineering, The Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China (S.D.); and CUHK
Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen 518057, China (L.J.)
Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi
network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGNindependent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal
immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles
(GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis
pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1
follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGNPM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we
have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1
for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent.
Plant development and growth require coordinated
tissue and cell polarization. Two of the most essential
cellular processes involved in polarization are cell expansion and cytokinesis, which determines cell morphology and functions (Jaillais and Gaude, 2008;
Dettmer and Friml, 2011; Li et al., 2012). Pollen tube and
root hair growth require highly polarized membrane
trafficking (Libault et al., 2010; Kroeger and Geitmann,
1
This work was supported by grants from the Research Grants
Council of Hong Kong (CUHK466011, 465112, 466613, CUHK2/CRF/
11G, C4011-14R, HKUST10/CRF/12R, and AoE/M-05/12), NSFC/
RGC (N_CUHK406/12), NSFC (31470294), Croucher-CAS Joint Lab,
and Shenzhen Peacock Project (KQTD201101) to L.J. This work was also
supported by NSFC (31570001) and Natural Science Foundation of
Guangdong Province (2016A030313401) to H.W., HKUST
VPRGO12SC02 grant to M.M.T.L., and by the grant HKUST12/
CRF/13G from the Research Grants Council of Hong Kong to M.M.
T.L., S.D., and L.J.
2
These authors contributed equally to the article.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Liwen Jiang ([email protected]).
H.W. and L.J. conceived the study; H.W. and X.Z. conducted most of
the research; H.W., X.Z., X.W., and A.H.Y.L. analyzed the data; H.W.,
T.Z., S.D., and M.M.T.L. performed STORM imaging and image reconstruction; H.W. and L.J. wrote the manuscript; L.J. supervised the project.
[OPEN]
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2012). Cytokinesis, by which new cells are formed,
separates daughter cells by forming a new structure
within the cytoplasm termed the cell plate (CP). Made
up of a cell wall (CW), surrounded by new plasma
membrane (PM), the cell plate is generally considered
to be an example of internal cell polarity in a nonpolarized plant cell (Bednarek and Falbel, 2002; Baluska
et al., 2006).
The conventional view of pollen tube tip growth and
cell plate formation is supported by polar exocytic
secretion of numerous vesicles (diameter of 60–
100 nm) to the pollen tube tip and phragmoplast areas
during cytokinesis. These polar exocytic vesicles,
which are generally believed to originate from the
Golgi apparatus, are delivered to the site of secretion
via the cytoskeleton and fuse with the target membrane with the aid of fusion factors (Jurgens, 2005;
Backues et al., 2007). However, whether these polar
exocytic vesicles undergoing post-Golgi trafficking are
part of the conventional Golgi-trans-Golgi network
(TGN)-PM/CP exocytosis or are derived from some
other unidentified exocytic secretion pathway remain
unclear.
Polar exocytosis is regulated and controlled by a
conserved Rho GTPase signaling network in fungi,
animals, and plants (Burkel et al., 2012; Ridley, 2013).
Rho of plant (ROP), the sole subfamily of Rho GTPases
in plant, participate in signaling pathways that regulate cytoskeleton organization and endomembrane
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Wang et al.
trafficking, consequently determining cell polarization,
polar growth and cell morphogenesis (Gu et al., 2005;
Lee et al., 2008). In growing pollen tubes, ROP1 participates in regulating polar exocytosis in the tip region via two downstream pathways to regulate apical
F-actin dynamics: RIC4-mediated F-actin polymerization and RIC3-mediated apical actin depolymerization.
A constitutively active mutant of ROP1 (CA-rop1)
prevents fusion of these vesicles with the PM and enhances the accumulation of exocytic vesicles in the apical cortex of pollen tubes (Lee et al., 2008). Although
ROP GTPases have been extensively researched, their
roles in polar membrane expansion in pollen tubes and
epidermal pavement cells remains unclear (Xu et al.,
2010; Yang and Lavagi, 2012), and there have been insufficient studies on the functions of ROPs in controlling cell plate formation during cytokinesis. Cell
division requires precise regulation and spatial organization of the cytoskeleton for delivery of secretion
vesicles to the expanding cell plate (Molendijk et al.,
2001).
In addition, newly made cell walls during cell expansion and cell plate formation require sufficient
plasticity in order to integrate new membrane materials
to support the polarized membrane extension. They
also should be strong enough to withstand the internal
turgor pressure and thereby maintain the shape of the
cell (Zonia and Munnik, 2011; Hepler et al., 2013). Recent studies have demonstrated that pectins are important for both cytokinesis and cell expansion (Moore
and Staehelin, 1988; Bosch et al., 2005; Chebli et al.,
2012; Altartouri and Geitmann, 2015; Bidhendi and
Geitmann, 2016). Pectins are one of the major cell wall
components of the middle lamella and primary cell
wall. They are polymerized and methylesterified in the
Golgi and subsequently released into the apoplastic
space as “soft” methylesterified polymers. The homogalacturonan components of pectin are later demethylesterified by pectin methylesterases (PMEs). The
demethylesterified pectins can be cross-linked, interact
with Ca2+, and finally form the “hard” pectin matrix of
the cell wall. Therefore, the enzymatic activity of PMEs
determines the rigidity of the cell wall (Micheli, 2001;
Peaucelle et al., 2011).
In Arabidopsis (Arabidopsis thaliana) and tobacco
(Nicotiana tabacum) pollen tubes, PMEs are found predominantly polar localized in the tip region and determine the rigidity of the apical cell wall (Bosch et al.,
2005; Jiang et al., 2005; Fayant et al., 2010; Chebli et al.,
2012; Wang et al., 2013). PME isoform knockout mutants in Arabidopsis (AtPPME1 or vanguard1) produce
unstable pollen tubes which burst when germinated
in vitro and have reduced fertilization abilities (Jiang
et al., 2005; Rockel et al., 2008). Recent studies have
shown that in growing tobacco pollen tubes, polar
targeting of NtPPME1 to the pollen tube apex depends
on an apical F-actin mesh network (Wang et al., 2013).
Although the functions of PME in cell wall constriction
are well documented, the intracellular secretion and
regulation mechanism of the exocytic process of PME
still remain largely unexplored. In addition, pectins are
also found to be abundant in the forming cell plate,
raising the possibility that PMEs may also function
during cell plate formation (Moore and Staehelin, 1988;
Dhonukshe et al., 2006).
In our study, we have used NtPPME1 as a marker to
identify a polar exocytic process which is distinct from
the conventional Golgi-TGN-PM exocytosis pathway
in both pollen tube tip growth and cell plate formation. We have identified a Golgi-derived secretory
vesicle (GDSV) for the polar secretion and targeting of
NtPPME1 to the cell wall that bypasses the TGN during
cell polarization. Further investigations using ROP1
mutants have shown that this polar exocytosis is ROP1
dependent.
RESULTS
Polar Exocytic Secretion of NtPPME1 to the Pollen
Tube Tip
Nicotiana tabacum pollen-specific pectin methylesterase (NtPPME1), which is regulated by apical F-actin,
plays a key role in pollen tube cell wall formation
(Bosch et al., 2005; Bosch and Hepler, 2006; Wang et al.,
2013). However, the details of post-Golgi intracellular
secretion of NtPPME1 remain unclear. To explore the
subcellular localization and to identify the endomembrane compartments mediating polar secretion of
NtPPME1, we first raised an antibody specific against
NtPPME1. The specificity the NtPPME1 antibodies was
initially tested via protein gel blot analysis. As shown
in Figure 1A, affinity-purified NtPPME1 antibodies
detected a major band at ;60 kD, the expected size of
NtPPME1, in total protein extracts from 2-h in vitro
germinated tobacco pollen tubes. As the background
control, preserum of NtPPME1 antibody was tested
and showed no detection in western, immunofluorescent labeling and immunoEM (Supplemental Fig. S1).
To further confirm the specificity of NtPPME1 antibody, pollen tubes expressing NtPPME1-GFP were
used for protein extraction and western blot analysis
using NtPPME1 and GFP antibodies. Both fusion
protein and endogenous proteins can be specifically
recognized (Supplemental Fig. S1A). Ideally, the specificity of the antibodies used in this study should be
further confirmed via using proper mutants in future
studies.
The tip-focused subcellular localization of NtPPME1
was shown via transient expression of NtPPME1-GFP
fusion protein in growing pollen tubes and confocal
immunofluorescence in fixed tobacco pollen tubes. As
shown in Figure 1B, the growing pollen tube expressing
NtPPME1-GFP via particle bombardment showed
punctate dots in the cytosol and on the surface of the
pollen tube tip. The NtPPME1 antibodies also labeled
the pollen tube apical region and punctate dots in the
cytosol (Fig. 1C). Such a distribution pattern is consistent with results obtained by transient expression of the
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Protein Trafficking in Plant Cell Wall Formation
Figure 1. Polar exocytosis of NtPPME1 in the pollen tube tip. A, Characterization of NtPPME1 antibodies with total protein from
2-h germinated tobacco pollen tubes. The asterisk indicates the expected size of NtPPME1. M, Molecular mass in kilodaltons. B,
Laser scanning of confocal microscopy (LSCM) image of a growing tobacco pollen tube expressing NtPPME1-GFP. C, Immunofluorescence labeling of NtPPME1 in tobacco pollen tube by using NtPPME1 antibodies. Scale bar = 25 mm. D, A representative
time-lapse image series of expressing NtPPME1-GFP during pollen tube tip expansion. The fluorescent signals of NtPPME1-GFP
remained tip-focused along with some intracellular punctate dots in the growing pollen tube. Scale bar = 25 mm.
NtPPME1-GFP fusion protein in pollen tubes. A representative time-lapse image series of expressing
NtPPME1-GFP in the growing pollen tube was shown
in Figure 1D. The fluorescent signals of NtPPME1-GFP
constantly remained on the pollen tube apex during the
tip expansion. In addition to the fluorescent signals
detected in the pollen tube tip, punctate signals were
also found within the cytoplasm of the pollen tubes
expressing NtPPME1-GFP. The cytoplasmic organelles
labeled by the NtPPME1-GFP were highly dynamic
and mobile, moving along with the cytoplasmic
streaming from distal to the tip region (Supplemental
Movie S1).
NtPPME1-GFP-Positive Compartments Are Distinct from
Conventional Post-Golgi Trafficking
Endosomal Compartments
To better characterize the nature of the NtPPME1GFP-positive organelle in growing pollen tubes, we
compared its localization with other post-Golgi trafficking organelle markers. As shown in Supplemental
Figure S2, A to E, the major signals of NtPPME1-GFP in
growing tobacco pollen tubes were observed to be
closely associated with, but not colocalized with, the
trans-Golgi marker (RFP-GONST1). They were also
separated from the PVC marker (RFP-AtVSR2), the
cis-Golgi marker (RFP-ManI), the TGN marker
(RFP-AtSCAMP4), and endocytic vesicles (FM4-64 dye
after 10-min uptake).
Next, we used pharmaceutical treatments to further
elucidate the identity of the NtPPME1-containing
compartments including the following: (1) Brefeldin A
(BFA), an ARF-GEF inhibitor, has been shown to induce
Golgi and TGN aggregations in various plant cell types
including pollen tubes and BY2 cells (Emans et al., 2002;
Robinson et al., 2008; Lam et al., 2009; Wang et al.,
2010a; Wang et al., 2011b); (2) wortmannin, a phosphoinositide 3-kinase inhibitor that causes homotypic
fusion of PVCs (Ethier and Madison, 2002; Tse et al.,
2004; Wang et al., 2009; Scheuring et al., 2011; Wang
et al., 2011b; Gao et al., 2014a; Zhao et al., 2015; Cui
et al., 2016); and (3) concanamycin A (ConcA), which is
a V-ATPase inhibitor and prevents post-TGN trafficking resulting in early endosome aggregations
(Robinson et al., 2004; Cai et al., 2011; Scheuring et al.,
2011). These drugs are extensively used and regarded
as useful tools in studying and dissecting endomembrane sorting and trafficking in eukaryotic cells. Although the apical localization of NtPPME1-GFP was
interrupted after the pharmaceutical treatments, which
might be due to pollen tube growth arrest and loss of
cell polarity, our results showed that NtPPME1-GFPpositive intracellular compartments were insensitive
to BFA, wortmannin, and ConcA treatments (Fig. 2, A–
D) when compared with the responses of the TGN
(GFP-AtSCAMP4) and PVC (GFP-AtVSR2) markers
(Fig. 2, E–J). GFP-AtSCAMP4 showed typical aggregations after 15-min BFA and ConcA treatments,
respectively, but remained punctate after 15-min
wortmannin treatment (Fig. 2, E–H). Quantifications of
the effects of drug treatment in pollen tubes are given in
Supplemental Figure S3. These results are consistent
with previous observations on the responses of the
TGN to BFA and ConcA treatments (Lam et al., 2007a;
Viotti et al., 2010; Wang et al., 2010a; Cai et al., 2011).
After 15-min wortmannin treatment, GFP-AtVSR2
showed the typical enlarged ring-like structures (Fig.
2, I and J). These results indicate that NtPPME1 are
delivered in a polar fashion to the pollen tube tip via a
different post-Golgi organelle, which is distinct from
the TGN and PVC.
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Wang et al.
Figure 2. NtPPME1-GFP-positve compartments are insensitive to pharmaceutical treatments in pollen tubes. A to
D, Growing pollen tubes expressing
NtPPME1-GFP were treated with 10 mg
mL21 BFA (B), 8.25 mM wortmannin (C),
and 2 mM ConcA (D) for 15 min respectively. E to H, Pollen tubes expressing
GFP-AtSCAMP4 (TGN marker) formed
aggregates after BFA and ConcA treatment for 15 min. I and J, Pollen tubes
expressing GFP-AtVSR2 (PVC marker)
were treated with wortmannin for 15 min
and formed enlarged ring-like structures
(J). Scale bar = 25 mm.
NtPPME1 Highlights the Forming Cell Plate during
Cytokinesis and Is Distinct from Conventional Post-Golgi
Trafficking Organelles in Tobacco BY2 Cells
Pectins are important to cell expansion during pollen
tube growth. In addition, recent studies have demonstrated that pectins are essential to cytokinesis, with
pectin demethylesterification being catalyzed by the
PMEs (Moore and Staehelin, 1988; Micheli, 2001; Bosch
et al., 2005; Dhonukshe et al., 2006; Peaucelle et al.,
2011). The forming cell plate is regarded as an example
of “inner cell polarity” in nonpolarized plant cells
(Jurgens, 2005; Backues et al., 2007; Mravec et al., 2011).
Although pectins are abundant in the forming cell plate,
how PMEs function at the cell plate remains unclear
(Micheli, 2001; Backues et al., 2007; Peaucelle et al.,
2011). NtPPME1 was highly expressed during cell plate
formation in growing pollen tubes (Supplemental Fig.
S4). Stable transgenic tobacco BY2 cells expressing
NtPPME1-GFP were generated as a prerequisite for
testing whether NtPPME1 is involved cell plate formation during cytokinesis. As shown in Figure 3A, the
NtPPME1-GFP signals accumulated in the forming
cell plate with some punctate dots in the cytosol
(Supplemental Movie S2). While in the nondividing
BY2 cells without polarity, the NtPPME1-GFP proteins
were delivered into the vacuole for degradation
(Fig. 3B). However, when compared with BY2 cells
expressing a TGN marker (GFP-AtSCAMP4), which
also highlights the forming cell plate, the dynamic responses of NtPPME1-GFP to different pharmaceutical
treatments were distinct from that of GFP-AtSCAMP4
in BY2 cells. In Figure 3, C to F, within 30 min of
treatments with BFA, ConcA, and wortmannin, respectively, the pattern of NtPPME1-GFP showed no
significant changes, while GFP-AtSCAMP4 formed
large aggregations after BFA and ConcA treatments
representing the typical responses of early endosome to
the treatment as previously reported (Lam et al., 2007a;
Viotti et al., 2010; Cai et al., 2011). Quantifications of the
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Protein Trafficking in Plant Cell Wall Formation
Figure 3. Polarity-associated NtPPME1 highlights cell plate during
cytokinesis and is insensitive to BFA, ConcA, and wortmannin in tobacco
BY2 cells. A and B, NtPPME1-GFP is polarity-associated and highlights
the forming cell plate during cytokinesis in transgenic tobacco BY2 cells
but degraded in the vacuole of a nondividing cell without polarity (B). C,
Transgenic tobacco BY2 cells expressing both NtPPME1-GFP and GFPAtSCAMP3 highlight cell plate during cytokinesis. D to F, Distinct
responses of transgenic tobacco BY2 cells expressing NtPPME1-GFP and
GFP-AtSCAMP3 to 10 mg mL21 BFA, 2 mM ConcA, or 8.25 mM wortmannin 30-min treatment. Scale bar = 50 mm. G and H, Super resolution
imaging by using STORM. BY2 cells were fixed and double-immunolabeled with NtPPME1 and OsSCAMP1 or AtEMP12 antibodies, respectively. Scale bar = 400 nm.
effects of drug treatment in BY2 cells are shown in
Supplemental Figure S3. Because of the 200 nm resolution limit of the conventional laser scanning confocal
microscopy, we performed superresolution imaging
using stochastic optical reconstruction microscopy
(STORM) to further confirm that NtPPME1-GFPpositive organelles are distinct from the TGN and
Golgi apparatus. BY2 cells were fixed and doubleimmunolabeled with NtPPME1 and OsSCAMP1 or
AtEMP12 antibodies. After image acquisition and reconstruction, NtPPME1-labeled cytosolic organelles
were separate from TGNs and the Golgi apparatus (Fig.
3, G and H).
In addition to the secretory activity at the cell plate
via the biosynthetic pathway, endocytic trafficking is
also directed and contributes to cell plate formation
(Dhonukshe et al., 2006; Boutte et al., 2010). The
endocytic tracer FM4-64 is incorporated into developing cell plates, suggesting that endocytosed material is delivered to the initiating and expanding cell
plates (Dhonukshe et al., 2006; Lam et al., 2008; Boutte
et al., 2010). However, the intracellular NtPPME1GFP-positive organelles were clearly separate from
endocytic endosomes, which were stained by FM4-64
after 10-min uptake in dividing BY2 cells (Fig. 4A1).
When the cells were treated with BFA, FM4-64 stained
endocytic vesicles formed large aggregates as previously reported (Robinson et al., 2008; Lam et al., 2009;
Wang et al., 2010a), but NtPPME1-GFP labeled organelles in the cytosol remained punctate and showed
no significant responses to BFA (Fig. 4A2). In contrast
to NtPPME1-GFP-positive, FM4-64-stained endocytic
vesicles fully colocalized with GFP-AtSCAMP4 in the
cytosol after 10-min uptake and both formed BFAinduced aggregates (Fig. 4A, 3 and 4). To identify
the sequential recruitments of endocytic and secretory materials during cell plate initiation, we next
performed time-lapse imaging of cell plate initiation
and traced the appearance of FM4-64 and NtPPME1GFP signals at the cell plate. As shown in Figure 4B,
a time course of images showed at the early stage of
cell plate initiation and formation, FM4-64 stained
endocytic vesicles reaching the cell plate prior to
NtPPME1-GFP. This result may suggests that membrane materials via endocytosis are first recruited for
cell plate membrane formation and afterward, the
cell wall modifying enzyme NtPME1 is subsequently secreted into the existing cell plate for cell
wall modification.
To further confirm the subcellular localization of
NtPPME1 in dividing BY2 cells, we re-examined its
subcellular distribution by confocal microscopy using
different antibodies against organelle markers in fixed
transgenic BY2 cells expressing NtPPME1-GFP.
NtPPME1-GFP signals were detected on cell plates
and some cytosolic punctate compartments, which
fully colocalized with the NtPPME1 antibody labeling
(Supplemental Fig. S5A). However, NtPPME1-GFPpositive cytosolic compartments were separate from
the TGN marker OsSCAMP1, the Golgi apparatus
marker AtEMP12, the clathrin-coated vesicle marker
CHC, the PVC marker VSR, the methylesterified pectin
marker JIM7, the de-methylesterified pectin marker
JIM5, and the cytokinesis-specific syntaxin KNOLLE
(Supplemental Fig. S5, B–H). Taken together, our results indicate that the NtPPME1-GFP-positive organelle mediates a polar protein secretion pathway from
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Wang et al.
Figure 4. Polar secretion of NtPPME
that contributes to cell plate formation
during cytokinesis is distinct from the
endocytic pathway in tobacco BY2 cells.
A, Polar secretion of NtPPME1 was not
overlapped with endocytic dye FM4-64 (1)
and insensitive to the 10 mg mL21 BFA
treatment (2). Transgenic tobacco BY2
cells expressing GFP-AtSCAMP3 used as a
control (3 and 4). Scale bar = 50 mm. B,
Time-lapse images of cell plate initiation
and formation in tobacco BY2 cell. (1)
NtPPME1-GFP on the forming cell plate.
(2) FM4-64-labeled endocytic membrane on the forming cell plate. Scale
bar = 75 mm. The ImageJ program with
the PSC colocalization plug-in (see
“Materials and Methods”) was used to
calculate the colocalizations between
these two fluorophores. Results are presented either as Pearson correlation coefficients or as Spearman’s rank correlation
coefficients, both of which produce r
values in the range 21 to 1, where 0 indicates no discernable correlation while +1
and 21 indicate strong positive or negative
correlations, respectively.
Golgi-to-CP, which is distinct from the classical GolgiTGN-CP pathway.
Immunogold EM Identification of Golgi-Derived Secretion
Vesicle as a Polarity-Associated Exocytic Vesicle for
NtPPME1 Secretion to Pollen Tube Tip and the Cell Plate
during Cytokinesis
To further morphologically characterize the
NtPPME1-GFP labeled intracellular compartments during pollen tube growth and cell plate formation, we
performed immunogold EM using the NtPPME1
antibody on ultrathin sections of high-pressure frozen/
freeze-substituted samples of 2-h germinated pollen
tubes and synchronized wild-type BY2 cells. An overview image of a pollen tube tip with a large number of
accumulated exo-/endocytic vesicles is shown in Figure
5A1. The NtPPME1 antibodies labeled the vesicles in the
pollen tube tip (Fig. 5A2). It is more challenging to preserve the pollen tube cell structures probably due to the
relevant dense cell contents and vigorous cytoplasmic
streaming. To better discern the fine structural details of
the pollen tube, high-pressure fixed pollen tubes were
subsequently treated with 2% osmium tetroxide during
the freeze substitution procedures. A representative
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Protein Trafficking in Plant Cell Wall Formation
structural image of the Golgi apparatus and TGN in a
pollen tube is given in Figure 5A3. A typical image of a
GDSV (;80 nm in diameter) was observed budding
from the transside of the Golgi (indicated by an arrow)
and separated from the TGN. Further immunogold
labeling with NtPPME1 antibodies revealed GDSV
(indicated by arrow), which was usually found budding
from the transside of Golgi stacks (Fig. 5A4). An
representative structural image of the TGN in pollen
tube labeled with OsSCAMP1 antibodies is shown in
Supplemental Figure S6A. Table I presents the
quantitative results from static images and indicates
that NtPPME1 is concentrated in the GDSVs budding
from the transside Golgi (38.7% of total account)
compared with the gold particles labeled on TGNs
(3.6% of total account). In contrast, OsSCAMP1 is
concentrated on TGNs (42.8% of total account), but
not on GDSVs (0.7% of total account) in the pollen
tube (Table I).
In dividing BY2 cells, GDSVs budding from the
transside of the Golgi were specifically labeled by
NtPPME1 antibodies (arrows) as shown in Figure 5B1,
but were absent from TGNs. Double immunogold EM
with NtPPME1 and OsSCAMP1 antibodies showed
GDSVs budding from the Golgi apparatus were labeled
by NtPPME1 antibodies, whereas the TGN was specifically recognized by OsSCAMP1 antibodies (Fig.
5B3). A representative structural image of the TGN in a
BY2 cell labeled with OsSCAMP1 antibodies is given in
Supplemental Figure S6B. In addition, the forming cell
plate in BY2 cells was also positively labeled with
NtPPME1 antibodies (arrows), as shown in Figure 5B2.
As expected, we found that NtPPME1-positive vesicles
(indicated by arrows) were distinct from clathrincoated vesicles (CCVs) labeled by CHC antibodies (indicated by arrowheads; Fig. 5B, 4–6). The distribution
of immunogold labeling with antibodies against
NtPPME1, OsSCAMP1, CHC, VSR, and AtEMP12 was
quantitated based on the static images (Table II). The
results obtained suggest that NtPPME1 resides on
GDSV, which is a distinct exocytic compartment from
Golgi apparatus, TGN, CCV, and multivesicular body
(MVB). Thus, these results indicate that GDSV serve as
a secretory organelle mediating the polar trafficking of
NtPPME1 directly from the Golgi apparatus to PM/CP
for cell wall formation during pollen tube tip expansion
and cell plate formation.
ROP1 Activity Controls NtPPME1 Polar Exocytic Secretion
and Targeting during Cell Plate Formation and Pollen
Tube Growth
ROP GTPase signaling plays a key role in terms of its
regulation of cell polarity, polar growth, and morphogenesis via the targeting of the cytoskeleton and vesicular trafficking in plant cells (Gu et al., 2005; Lee
et al., 2008; Fu et al., 2009). To explore the mechanism
that regulates GDSV-mediated NtPPME1 polar trafficking in pollen tube growth and cell plate formation,
we tested the functions of ROP1 in regulating NtPPME1
localization and targeting. Transgenic BY2 cells
expressing NtPPME1-GFP were synchronized and
subsequently bombarded with RFP-CA-rop1 and RFPDN-rop1, respectively (Supplemental Fig. S7). After
incubation for 12 h, fluorescence recovery after photobleaching (FRAP) analysis studying the roles of ROP1
in exocytic targeting of NtPPME1-GFP to the cell plate
was performed. A section of the whole cell plate was
photobleached, and the recovery of NtPPME1-GFP
fluorescence in the bleached region was monitored
every 5 s. We reasoned that the recovery was caused by
polar secretory activity to the cell plate. An example of
the FRAP analysis of a BY2 cell expressing NtPPME1GFP is shown in Figure 6A (top). Prior to photobleaching, NtPPME1-GFP appeared as a bright line in
the middle of the BY2 cell (Fig. 6A, top). After photobleaching, the fluorescence signal in the cell plate area
was reduced to ;3% of its original level (highlighted by
the white cycle in Figure 6A, top). GFP signals started to
reappear in the cell plate region within 45 s, reaching
.85% of their original intensity 540 s after bleaching.
The half-time of fluorescence recovery in the cell plate
area was ;270 s (n = 4; Fig. 6C). The recovery of the GFP
signal took place first at both edges of the cell plate and
then gradually moved toward the center (Fig. 6A, top;
Supplemental Movie S3). Furthermore, BY2 cells
expressing NtPPME1-GFP coexpressed with CA-rop1
or DN-rop1 exhibited a different FRAP rate and pattern. CA-rop1 significantly increased the rate of fluorescence recovery. As shown in Figure 6A (middle), the
GFP signals started to reappear in the cell plate region
within 15 s, but the recovery of GFP signal intensity in
the bleached cell plate region reached only ;40% of its
original level 540 s after photobleaching (n = 4; Fig. 6C;
Supplemental Movie S4). In contrast, DN-rop1 strongly
Table I. Distribution of immunogold labeling with antibodies against NtPPME1 and OsSCAMP1 of
tobacco pollen tubes
To elucidate the distribution of the NtPPME1 antigens in GDSV, which is distinct from OsSCAMP1
antigen in the TGN, photographs of GDSV with distinguishable TGN, Golgi apparatus, endoplasmic
reticulum (ER), and MVB were taken and the number of gold particles were counted for each individual
organelle. Fifty-three GDSVs from different fixations of tobacco pollen tubes were evaluated for the label.
Gp, Number of gold particles.
Antibody
GDSV
Tip Vesicle
TGN
Golgi
ER
MVB
Gp (%)
NtPPME1
OsSCAMP1
175(38.7)
5 (0.7)
227 (50.3)
298 (47)
16 (3.6)
217 (42.8)
15 (3.1)
20 (3.2)
12 (2.6)
17 (2.7)
7 (1.7)
22 (3.5)
452 (100)
579 (100)
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Wang et al.
Figure 5. NtPPME1-defined GDSVs, independent from TGN and CCVs, contribute to pollen tube tip growth and cell plate formation
in tobacco BY2 cells. High-pressure frozen/freeze-substituted samples of 2-h germinated pollen tubes and synchronized wild-type
BY2 cells were used for immunolabeling. A, Pollen tubes were immunolabeled with NtPPME1 antibodies. (1) Overview of the ultrastructure of a tobacco pollen tube tip. (2) NtPPME1-labeled exocytic secretory vesicles accumulated in the pollen tube tip region. (3)
Ultrastructure of secretory vesicle which is directly derived from Golgi apparatus and is distinct from the TGN. (4) NtPPME1 labeled
the GDSVs near the Golgi apparatus. B, ImmunoEM of synchronized BY2 cells. (1) NtPPME1 antibodies specifically labeled GDSVs
(arrows), but not TGN or TGN-derived CCVs. (2) NtPPME1 antibody labeled the cell plate in BY2 cells (arrows). (3) BY2 cells were
double-labeled with NtPPME1 and TGN marker OsSCAMP1 antibodies. NtPPME1-labeled GDSVs (10 nm, indicated by arrows)
were distinct from TGN, which was labeled by OsSCAMP1 antibody (15 nm, indicated by arrowheads). (4) NtPPME1 antibody
labeled secretion vesicles on cell plate (10 nm, arrows) were separate from CHC antibody-labeled CCVs on or near the cell plate
(6 nm, arrowheads). 5 and 6 are enlarged areas from 4.
inhibited the rate of fluorescence recovery (Fig. 6A,
bottom). The GFP signals started to reappear in the cell
plate region only after 1080 s. In addition, the recovery
of GFP signal intensity in the bleached cell plate region
reached only ;10% of its original level 540 s after
photobleaching (n = 4; Fig. 6C; Supplemental Movie
S5). To better illustrate the role of ROP1 in controlling
the polar targeting of NtPPME1 to the cell plate, GFP
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Protein Trafficking in Plant Cell Wall Formation
Table II. Distribution of immunogold labeling with antibodies against NtPPME1, OsSCAMP1, CHC, VSR,
and AtEMP12 in BY2 cells
To elucidate the distribution of the NtPPME1 antigens in the GDSV, photographs of GDSV with
distinguishable TGN, CCV, Golgi apparatus, and MVB were taken and the number of gold particles was
counted for each individual organelle. Forty-four GDSVs from different fixations of BY2 cells were
evaluated for the label. Gp, Number of gold particles; CHC, Clathrin H chain.
Antibody
NtPPME1
OsCAMP1
CHC
VSR
AtEMP12
GDSV
139
14
3
1
2
(38.2)
(3.6)
(1.7)
(1.5)
(3.2)
TGN
13
110
63
2
6
(3.6)
(27.9)
(35.2)
(3.0)
(9.5)
CCV
8
98
90
1
1
(2.2)
(24.9)
(50.3)
(1.5)
(1.6)
signal intensities of the cell plate region after photobleaching were measured and displayed at 180 s from
two directions, along the cell plate (indicated with a
white line) and across the cell plate (indicated with a red
line) as shown in Figure 6B. The cell plate started to
recover, and the recovery pattern was from the two
edges of the cell plate and then gradually moved to the
central region (Fig. 6B1). CA-rop1 disrupted the GFP
signal sequential recovery pattern, and the cell plate
signal was partially recovered (Fig. 6B2). DN-rop1
strongly inhibited the GFP signal recovery on the cell
plate (Fig. 6B3). To further demonstrate that the polar
exocytosis and targeting of NtPPME1 to cell plate is
ROP1-dependent, we treated BY2 cells expressing
NtPPME1-GFP with 50 mM catechin-derivative
(2)-epigallocatechin gallate (EGCG), a PME inhibitor
(Lewis et al., 2008; Wolf et al., 2012), or coexpressed
with CA-rop1 or DN-rop1, followed by confocal
imaging. As shown in Supplemental Figure S8, the cell
plate formation was strongly inhibited by treatment of
the PME activity inhibitor EGCG, as well as by coexpression of DN-rop1 or CA-rop1.
Furthermore, ROP1 also controls the polar targeting
of NtPPME1-GFP to the pollen tube tip and pollen tube
growth (Fig. 6D). Expression of CA-rop1 disrupted the
apical localization of NtPPME1-GFP, causing the pollen
tube tip to swell and inhibiting pollen tube growth (Fig.
6D2). Expression of DN-rop1 abolished the apical localization of NtPPME1-GFP and inhibits pollen tube
growth (Fig. 6D3). We also examined the effects of expression CA-rop1 and CA-rop1 at earlier stage of
pollen tubes expressing NtPPME1-GFP. As shown in
Supplemental Figure S9, expression of CA-rop1 or
CA-rop1 abolished NtPPME1-GFP apical localization
and strongly inhibited, but not completely stopped,
pollen tube germination and growth (Supplemental
Fig. S9). Taken together, these results suggest that
ROP1 affects the polar targeting of NtPPME1 to the
forming cell plate and growing pollen tube tip, which
in turn regulates cell plate formation and pollen tube
growth.
The exocytic targeting of SCAMP to PM has previously been shown to be achieved via the conventional
Golgi-TGN-PM exocytosis pathway (Cai et al., 2011).
Here we further tested the roles of ROP1 in controlling
CP
171
127
15
3
4
(47.0)
(32.2)
(25.7)
(4.5)
(6.3)
Golgi
24
31
3
3
44
(6.6)
(7.9)
(1.68)
(4.5)
(69.8)
MVB
9
14
5
56
6
(2.5)
(3.6)
(2.79)
(84.8)
(9.5)
Gp (%)
364
394
179
66
63
(100)
(100)
(100)
(100)
(100)
the polar targeting of SCAMP to PM in pollen tube
(Supplemental Fig. S10). Our results suggest that ROP1
also participates in regulating the polar targeting of
SCAMP by the conventional exocytosis pathway via
TGN. Thus, it indicates that ROP1 may function as a
master regulator for both conventional TGN-passing
and GDSV-mediated exocytosis pathways in pollen
tube growth and cell plate formation.
DISCUSSION
NtPPME1 Resides on a Polarity-Associated
Exocytic Vesicle
In plants, pectin modifications by PMEs are usually
accompanied with changes in plasticity, pH, and ionic
contents of the cell wall, and finally influence plant cell
growth and development (Micheli, 2001; Peaucelle et al.,
2011). Previous studies indicate that NtPPME1 plays
essential roles in pollen tube apical cell wall formation by
determining its rigidity via demethylesterification of
pectins (Bosch et al., 2005; Fayant et al., 2010; Chebli
et al., 2012; Wang et al., 2013; Bidhendi and Geitmann,
2016). However, the subcellular localization and polar
exocytosis of NtPPME1 remains unexplored. In addition, although pectins are also found to be abundant in
the forming cell plate during cytokinesis, the involvement and functions of PMEs in the cell plate formation remain to be characterized (Baluska et al., 2005;
Dhonukshe et al., 2006).
In this study, transgenic tobacco BY2 cells expressing
NtPPME1-GFP were generated. The functional involvement and localization of NtPPME1 in cytokinesis
was examined. The subcellular localization and dynamics of NtPPME1 in growing pollen tubes and cell
plate formation (Figs. 1 and 3) suggest that the exocytosis and targeting of NtPPME1 for cell wall formation
is closely associated with cell polarity in both cell expansion and cytokinesis. Further characterization of the
NtPPME1 punctae in the cytoplasm of pollen tubes and
dividing BY2 cells shows that NtPPME1 resides on a
polar exocytic vesicle directly derived from transside
Golgi (Fig. 5). GDSVs have previously been morphologically detected by 3D tomogram reconstruction and
modeling (Kang et al., 2011; Gao et al., 2014b).
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Wang et al.
Figure 6. ROP1 regulates NtPPME1 polar exocytic secretion and targeting in cell wall formation during cytokinesis and pollen
tube growth. A, FRAP analysis of NtPPME1-GFP highlighted cell plate formation (top) or coexpressed with CA-rop1 (middle) or
DN-rop1 (bottom) during cytokinesis in tobacco BY2 cells. A series of images were shown before bleaching, immediately after
bleaching (0 s), and then recovery of fluorescence was shown after bleaching at the indicated time points. Numbers indicate the
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Protein Trafficking in Plant Cell Wall Formation
However, little is known about their biological functions in mediating protein trafficking. In our study, we
have used NtPPME1 as a marker to reveal that GDSV
mediates polar exocytosis of NtPPME1 for the cell
wall formation. This is supported by several lines of
evidence: (1) Both confocal microscopy and superresolution imaging have shown that the NtPPME1GFP-positive intracellular compartment is distinct
from various endosomal markers involved in the conventional Golgi-TGN-PM exocytosis (Figs. 3 and 4; see
Supplemental Figs. S2 and S5). (2) Pharmaceutical
treatments with BFA, wortmannin, and ConcA have
been well-established and are regularly used as a tool
to influence conventional secretion, degradation, or
endocytic organelles/vesicles in previous studies
(Tse et al., 2004; Robinson et al., 2008; Lam et al., 2009;
Cai et al., 2011; Scheuring et al., 2011; Gao et al., 2015;
Cui et al., 2016). Drug treatments of the pollen tubes
and BY2 cells expressing NtPPME1-GFP showed no
obvious effects on NtPPME1-GFP labeled intracellular
compartments except for disruption of the apical localization of NtPPME1 in pollen tube, which is likely to
be caused by pollen tube growth arrest and cell polarity
lost after the treatments (Fig. 2; Supplemental Fig. S3).
In addition, the distinct responses and subcellular localizations between NtPPME1 and SCAMP after BFA
and ConcA treatments also rule out the possibility that
the intracellular NtPPME1-positive compartments
might be derived from different functional domains
of TGN or TGN-derived exocytic vesicles (Figs. 2, 3,
and 4). (3) Ultrastructural analysis by immunogold EM
with NtPPME1 antibodies have demonstrated that
NtPPME1 resides on GDSVs that are distinct from the
TGN in both pollen tube and tobacco BY2 cells (Fig. 5).
Taken together, our results demonstrate that the polar
exocytosis of NtPPME1 for cell wall rigidity modifications is mediated by a Golgi directly derived transport
compartment, GDSV, in pollen tube growth and cell
plate formation.
Polar Cellular Trafficking of NtPPME1 Highlights
Golgi-PM Exocytosis Pathway Bypassing the TGN
It is generally considered that exocytic vesicles originate from the Golgi as most of the cell wall formation
materials like pectins are first methlyesterified in the
Golgi first and are then delivered to the pollen tube tip
to meet the requirements of cell wall expansion (Carter
et al., 2004; Lam et al., 2007b; Bove et al., 2008; Cai and
Cresti, 2009; Hepler et al., 2013). However, whether
these exocytic vesicles are solely derived from conventional Golgi-TGN-PM exocytic pathway or are
possibly derived from other, unconventional, exocytic
processes remains unknown.
Unconventional protein secretion (UPS) pathways
are known and well-documented in yeast and mammalian cells, but recent studies suggest that UPS also
exists in plant cells (Ding et al., 2012; Robinson et al.,
2016). Although it is not known exactly how many
different types of UPS pathways exist in plant cells,
studies of exocyst-positive organelle (EXPO)-mediated
UPS indicate that multiple exocytosis pathways may
possibly co-operate and compensate for the classical
endoplasmic reticulum-Golgi-TGN-PM protein exocytosis pathway (Wang et al., 2010b). Additionally, recent
studies have shown the existence of different populations of exocyst-positive organelles mediating multiple
post-Golgi transport routes from Golgi apparatus or
TGN to PM/CW (Crowell et al., 2009; De Caroli et al.,
2011; Boutte et al., 2013; McFarlane et al., 2013; Poulsen
et al., 2014). Our results suggests that polar exocytosis of NtPPME1 along the Golgi-to-PM/CP pathway
bypassing TGN, may complement the conventional
Golgi-TGN-PM secretion pathway as with UPS. Our
findings thus support that NtPPME1 is exocytotically
secreted to the PM directly from trans-Golgi via GDSV.
It will be technically challenging in future research to
find out if the NtPPME1 secretion might involve other
intermediate compartments or if NtPPME1 transiently
and rapidly passes through TGN. Moreover, previous
studies have shown that SCAMP is localized on the
tubular-vesicular structures TGN/EE and its postGolgi trafficking is clathrin-dependent (Lam et al.,
2007a). SCAMP has been well-defined and used as a
TGN maker in addition to its PM localization (Lam
et al., 2007a; Lam et al., 2008; Lam et al., 2009; Wang
et al., 2010a; Cai et al., 2011; Gao et al., 2012). Interestingly, Toyooka et al. (2009) described the secretory
vesicle cluster (SVC), which is derived from TGN and
consists of a cluster of vesicles often near TGN, as a
distinct secretory compartment mediating targeting of
Figure 6. (Continued.)
order of signal recovery. Scale bar = 50 mm. B, Fluorescence-intensity recovery analysis of images in transgenic tobacco BY2 cell
expressing NtPPME1-GFP (1), or coexpressed with CA-rop1 (2) or DN-rop1 (3) at 180 s after bleaching. The corresponding
fluorescence intensity recovery along the cell plate (white line) was shown in the middle column and the fluorescence intensity
recovery across the cell plate (red line) was indicated on the third column. C, Quantitative analysis of FRAP for A. Fluorescence
recovery was measured by calculating the mean GFP signal intensity on the cell plate of the region of interest. For the top, fluorescence of the cell plate area recovered completely in 540 s, with a 270 6 10 s half-time of recovery (▪). For the middle,
fluorescence at the cell plate area recovered dramatically without sequential order in 120 s, with a 60 6 10 s half-time of recovery
( ). The fluorescent intensity of recovered cell plate is lower than that of the NtPPME1-GFP-highlighted cell plate alone. In
contrast, little fluorescence recovery occurred in the cell plate area when coexpresssed with DN-rop1 (c). Similar results were
obtained from four individual experiments. D, Confocal images showing the apical targeting of NtPPME1-GFP was inhibited
when coexpressed with CA-rop1 (2) or DN-rop1 (3) in growing pollen tube. Scale bar = 25 mm.
▴
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Wang et al.
SCAMP2 to PM. GDSVs are morphologically different
from SVCs and are usually found near the trans-Golgi
without clustering (Fig. 5). GDSVs mediate intracellular
trafficking and targeting of NtPPME1 but are different
from the secretory vesicles, which were labeled by JIM5
and JIM7 (Supplemental Fig. S5), whereas SVC contains
cell wall components such as JIM7. These results also
support the previous hypothesis that pectin and PME
are packed and secreted by different intracellular vesicles so as to avoid earlier interactions between the enzyme and it substrate (Micheli, 2001). Recent studies
show that cell plate formation also needs cell surface
material, including cell wall components, which is
rapidly delivered to the forming cell plate via endocytosis (Dhonukshe et al., 2006; Kim and Brandizzi, 2014).
Therefore, JIM5 labeled intracellular compartments
containing de-methylesterified pectins are likely endocytic in nature, seperate from the NtPPME1-GFPpositive exocytic compartments.
SCAMP also highlights the forming cell plate during
cytokinesis and colocalized with the internalized
endosomal marker FM4-64 on the forming cell plate
during cell division (Lam et al., 2007a, 2007b; Lam
et al., 2008). However, when compared with GFPAtSCAMP3 and the endocytic endosomal marker
FM4-64, NtPPME1-GFP punctae in pollen tubes and
dividing BY2 cells are distinct from the classical TGNs/
EEs, which usually bear clathrin coats (Figs. 2, 3, and 4).
Furthermore, these NtPPME1-GFP-positive cytosolic
punctae are insensitive to most pharmaceutical treatments, which affect either the secretion, recycling or
endocytic pathways.
During cytokinesis, plant cells require specific proteins (e.g. KNOLLE and Rab GTPase) to guard the polar
delivery, docking and fusion of vesicles carrying wall
materials to the forming cell plate (Lukowitz et al.,
1996). Meanwhile, organelles especially the Golgi apparatus relocate near the cell plate to aid secretion. Interestingly, we found that the signals of NtPPME1-GFP
on the cell plate during cytokinesis are separated from
cytokinesis-specific syntaxin KNOLLE, which is
reported to be BFA sensitive (Boutte et al., 2010;
Supplemental Fig. S5B8). This may indicate that cell
plate formation may need specific types of secretion
vesicles like GSDVs as the complemental exocytosis
pathway.
ROP1 Regulates Polar Exocytosis of NtPPME1
In trying to understand the underlying mechanisms
of the polarity-associated exocytosis of NtPPME1 in cell
wall formation during cell expansion and cell cytokinesis, we tested and found that ROP1 may function as
an essential regulator in controlling GDSV-mediated
polar targeting of NtPPME1 to the pollen tube tip and
cell plate (Fig. 6). The behavior and function of the
highly dynamic populations of apical F-actin, which
localizes to the tip region of the pollen tube, are regulated by ROP1 signaling network. CA-rop1 will
enhance the accumulation of exocytic vesicles to the
apical cortex of pollen tubes, leading to balloon-shaped
tip and resulting in cell polarity lost. Expression of
DN-rop1 can promote disassembly of F-actin and prevent polar exocytic vesicle transport and fusion with
the apical PM (Lee et al., 2008; Fu et al., 2009). However,
the origins and identities of these polar exocytic vesicles
regulated by ROP1 remain elusive.
We previously demonstrated that the tip localization
of NtPPME1 in the growing pollen tube is apical F-actin
dependent. Disruption of apical F-actin organization
and structures will abolish the tip localization of
NtPPME1 and alter the cell wall rigidity (Wang et al.,
2013). In this study, we found that ROP1 acts as an
essential regulator in controlling the polar exocytosis of
GDSV-mediated NtPPME1 and its targeting to the apical PM in growing pollen tubes. Expression of either
CA-rop1 or DN-rop1 abolished the tip localization of
NtPPME1-GFP and strongly inhibited pollen tube
growth (Fig. 6D). NtPPME1-GFP is strictly focused on
the pollen tube tip, and its expression intensity is closely
associated with pollen tube polarity and growth oscillation (Wang et al., 2013). In addition to the GDSVmediated NtPPME1 polar exocytosis pathway, the
conventional Golgi-TGN-PM exocytic process, which
mediates SCAMP secretion and targeting to PM, was
also found to be controlled by ROP1 (Supplemental Fig.
S10).
Besides the roles of ROP1 in controlling pollen tube
growth, recent studies suggest ROPs might also participate in other cell polarization processes (Yang and
Lavagi, 2012). Cell plate formation in plant cells is
assisted by the insertion of plant-specific polarized
proteins/membrane to the forming sites (Bednarek and
Falbel, 2002; Jurgens, 2005; Backues et al., 2007). However, evidence for a function of ROPs in cell plate formation has not been reported, although overexpression
of fluorescence-labeled ROP4 shows a localization at
the forming cell plate (Molendijk et al., 2001).
By using FRAP analysis, we have shown that
NtPPME1-mediated polar secretion and fusion with the
cell plate is highly active at the growing edges of the cell
plate and the NtPPME1-GFP signals recovered from the
two edges sequentially to the central region of the
forming cell plate. Interestingly, although CA-rop1 expression induced depolarization of cell plate signal recovery, it greatly accelerated the rate of recovery of
NtPPME1 to the cell plate. In contrast, DN-rop1 expression significantly suppressed the polar secretion
and targeting of NtPPME1 to the forming cell plate (Fig.
6, A–C). Therefore, our data provides a direct support
for a crucial role of ROP1 in regulating polar exocytosis
and targeting of GDSVs to the forming cell plate during
cytokinesis. This further confirms a more general master role of ROP1 in the regulation of plant cell polarity.
In summary, we have identified an alternative Golgito-PM polar exocytosis via the GDSV, which mediates
the polar secretion of NtPPME1 to the apoplast for cell
wall rigidity modification during pollen tube expansion
and cell plate formation. Several important questions
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Protein Trafficking in Plant Cell Wall Formation
will need to be addressed in future studies. For example,
what is the biological significance of GDSV-mediated
exocytosis? What are the molecular characteristics of
GDSVs? How is PPME1 specifically sequestered in
GDSVs rather than being included in secretory vesicles?
What are its up-/down-regulatory effectors? Using a
combination of biochemical, proteomic, cellular, and
genetic approaches to address these questions in future
studies, we will be able to understand more about
this GDSV-mediated secretion pathway as well as its
significance to cell polar morphogenesis and plant
development.
MATERIALS AND METHODS
Plant Materials and Pollen Tube Germination
Tobacco (Nicotiana tabacum) plants were grown in the greenhouse at 22°C
under a light cycle of 12 h light and 12 h dark. Fresh pollen grains were harvested before use. For pollen tube germination, pollen grains were suspended
in tobacco pollen-specific germination medium containing 10% Suc, 0.01% boric
acid, 1 mM CaCl2, 1 mM Ca(NO4) 2∙H2O, 1 mM MgSO4∙7H2O (pH 6.5) at 27.5°C
for 2 h.
Particle Bombardment of Tobacco Pollens and BY2 Cells
For tobacco pollens, fresh anthers were harvested from 10 to 12 tobacco
flowers and transferred into 20 mL of pollen germination medium. They were
vortexed vigorously to release pollen grains into the germination medium.
Pollen grain preparation and subsequent transient expression of proteins in the
growing pollen tube via particle bombardment were carried out as previously
described (Wang and Jiang, 2011a). For BY2 cells, in order to increase the expression efficiency in dividing BY2 cells, cell synchronization was first performed as previously described (Kumagai-Sano et al., 2006). The synchronized
BY2 cells were then vacuum-filtered onto a filter paper and placed in the center
of a Petri dish with BY2 culture agar medium. The sequential procedures for
gene delivery into BY2 cells via particle bombardment were the same with the
steps for pollen tube bombardment. The bombarded cells were kept in dark in
plant growth chamber (27°C) for 8 to 12 h prior to observation for fluorescent
signals.
Electron Microscopy of the Resin-Embedded Cells
The general procedures for TEM sample preparation and ultra-thin sectioning of samples were as previously described (Tse et al., 2004). For pollen
tubes and BY2 cells, high-pressure freezing and substitution were carried out as
previously described (Wang et al., 2013). Subsequent freeze substitution was
carried out in dry acetone containing 0.1% uranyl acetate at 285°C. Infiltration
with HM20, embedding, and UV polymerization were performed stepwise
at 235°C. For immunolabeling, standard procedures were performed as described previously (Cui et al., 2014). The working concentration of affinitypurified antibodies is 40 mg/mL. Gold particle-coupled secondary antibodies
were diluted 1:50, followed by the poststaining procedure by aqueous uranyl
acetate/lead citrate. Ultrathin sections were examined using a Hitachi H-7650
transmission electron microscope with a CCD camera operating at 80 kV
(Hitachi High-Technologies Corporation).
Immunofluorescence Labeling and Confocal Imaging
Fixation and preparation of BY2 cells/pollen tubes and subsequent labeling
with antibodies and analysis by immunofluorescence were performed as previously described (Tse et al., 2004; Zhuang and Jiang, 2014). In general, the
confocal fluorescence images were collected using a Leica TCS SP8 system with
the following parameters: 633 water objective, 23 zoom, 900 gain, 0 background, 0.168 mm pixel size, and photomultiplier tubes detector. The images
from pollen tubes labeled with antibodies were collected with a laser level of #
3% to ensure that the fluorescent signal was within the linear range of detection
(typically 0.5% or 1% laser was used). Colocalizations between two fluorophores were calculated by using ImageJ program with the PSC colocalization
plug-in (French et al., 2008). Results are presented either as Pearson correlation
coefficients or as Spearman’s rank correlation coefficients, both of which
produce r values in the range (21 to 1), where 0 indicates no discernable correlation while +1 and 21 indicate strong positive and negative correlations,
respectively.
Superresolution Microscopy Imaging by STORM
The two-channel STORM was built upon a Nikon Ti-E inverted microscope
basing with perfect focus system. Laser excitations for Alexa 647 channel and
Alexa 750 channel were provided by a 656.5 nm DPSS laser and a 750 nm diode
laser, respectively. The fluorescent signals of both channels were collected by a
1003 TIRF objective (Nikon) and passed through a home-built channel splitter
before simultaneously forming images on an EMCCD (Andor, IXon-Ultra). One
emission filter was placed in each path of the channel to block the excitation
laser. To stabilize the sample, the perfect focus system was used to lock the
focus, and the sample drift was measured with the brightfield image of cells via
a third channel and then was compensated in real time by an x-y piezo stage
(PIP545.R7.XYZ). All measurement, control, and acquisition were done using
custom written software. Prior to STORM imaging, the desired position is located using conventional fluorescence image with relatively low laser excitation
power, typically 60W/cm2 for a 656.5 nm laser in an Alexa 647 channel and
80W/cm2 for a 750 nm laser in an Alexa 750 nm channel. During the STORM
acquisition, the laser power was raised to 2kW/cm2 and 4.5kW/cm2, respectively. Blinking signals of both channels were recorded simultaneously using
EMCCD with 1003 EM gain at 30 frames per second for 30,000 frames. The data
were then analyzed by using Gaussian fitting software to reconstruct the
superresolution image. Throughout STORM experiments, the highly inclined
and laminated optical sheet geometry (Tokunaga et al., 2008; Wang, 2016) was
applied so that the illuminated area in cell is confined to a 3 mm thick sheet
10 mm above the coverslip, thus effectively reducing the background caused by
out-of-focus signal. All cells for STORM imaging were immobilized on poly-Lys
coated coverslip and mounted in STORM imaging buffer: 200 mM Tris-Cl (pH
9.0; Sigma-Aldrich, T2819) containing 10% (w/v) Glc (Sigma-Aldrich, G8270),
0.56 mg/mL Glc oxidase (Sigma-Aldrich, G2133), 57 mg/mL catalase (SigmaAldrich, C3515), 2 mM cyclooctatetraene (TCI, C0505, dissolved in DMSO),
25 mM TCEP (Sigma, 646547), 1 mM ascorbic acid (TCI, A0537), and 1 mM
Methyl Viologen (Sigma-Aldrich, 856177).
Drug Treatment and Antibodies
Stock solutions of wortmannin (1 mM in DMSO; Sigma-Aldrich), BFA (1 mM
in DMSO; Sigma-Aldrich), FM4-64 (4 mM in DMSO; Invitrogen), ConcA (1 mM
in DMSO; Sigma-Aldrich), and catechin-derivative (2)-EGCG (5 mM in H2O,
Sigma-Aldrich) were prepared. These drugs were diluted in germination medium to appropriate working concentrations before incubation with germinating pollen tubes. For each drug treatment, germinating pollen tubes or BY2
cells were mixed with the drugs in working solutions in the medium at a 1:1
ratio to minimize sample variation, and such a method of drug treatment has
been well-documented with success in both pollen tubes and BY2 cells of previous studies (Tse et al., 2004; Tse et al., 2006; Robinson et al., 2008; Lam et al.,
2009; Wang et al., 2010a; Cai et al., 2011; Wang et al., 2011b; Zhuang et al., 2013;
Cui et al., 2014; Ding et al., 2014; Gao et al., 2014b; Gao et al., 2015; Shen et al.,
2016; Wang et al., 2016; Chung et al., 2016). The JIM5 and JIM7 antibodies were
purchased from the Paul Knox Cell Wall Lab at the University of Leeds (Plant
710Probes).
A synthetic peptide (CQPTPYKQTCEKTLSSAKNASEPKDF) within the N
terminus of tobacco NtPPME1 was synthesized (GenScript) and used as antigen
to raise the antibody. The synthetic peptide was conjugated with keyhole limpet
hemocyanin and used to immunize nine rats at the animal house of the Chinese
University of Hong Kong (CUHK). The generated antibodies (anti-NtPPME1)
were further affinity-purified with a CnBr-activated Sepharose (Sigma-Aldrich;
catalog No. C9142-15G) column conjugated with synthetic peptides as described
previously (Jiang and Rogers, 1998). AtVSR1, OsSCAMP1, and AtEMP12 antibodies were generated as previously described (Tse et al., 2004; Lam et al.,
2007a; Gao et al., 2012). The JIM5, JIM7, and LM7 antibodies were purchased
from the Paul Knox Cell Wall Lab at the University of Leeds (PlantProbes). A
monoclonal antibody against CHC (at 250 mg/mL) was purchased from BD
Bioscience (catalog no. C610500). A polyclonal antibody against KNOLLE was
purchased from Agrisera AB (catalog no. AS06168).
Plant Physiol. Vol. 172, 2016
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Copyright © 2016 American Society of Plant Biologists. All rights reserved.
Wang et al.
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers NtPPME1(AY772945), AtVSR2
(NM_128582), AtSCAMP3 (At2g20840), and AtSCAMP4 (At1g03550).
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Characterization of the NtPPME1 antibody.
Supplemental Figure S2. NtPPME1-GFP-positive compartments are distinct from PVC, endocytic vesicle, TGN, and Golgi makers in growing
pollen tubes.
Supplemental Figure S3. Quantification analysis of cell responses to the
drug treatments.
Supplemental Figure S4. NtPPME1 expression analysis in pollens and BY2
cells.
Supplemental Figure S5. NtPPME1-positive compartments are distinct
from different organelle markers in tobacco BY2 Cells.
Supplemental Figure S6. Immunogold electron microscopy localization of
SCAMP1 in pollen tubes and BY2 cells.
Supplemental Figure S7. Coexpression of NtPPME1-GFP with RFP-CArop1 or RFP-DN-rop1 in tobacco BY2 cells during cytokinesis.
Supplemental Figure S8. The effects of inhibition of PME activity and
expression of CA-rop1 or DN-rop1 on NtPPME1-GFP polar exocytic
targeting to the forming cell plate.
Supplemental Figure S9. Coexpression of NtPPME1-GFP with CA-rop1 or
DN-rop1 in the early stages of pollen tube germination.
Supplemental Figure S10. FRAP of GFP-AtSCAMP4-stained apical PM in
tobacco pollen tubes.
Supplemental Movie S1. Dynamics of NtPPME1-GFP in a growing pollen tube.
Supplemental Movie S2. NtPPME1-GFP highlights the forming cell plate
in a dividing transgenic BY2 cell.
Supplemental Movie S3. FRAP analysis of NtPPME1-GFP at cell plate
formation in BY2 cells.
Supplemental Movie S4. FRAP analysis of NtPPME1-GFP at cell plate
formation in BY2 cells coexpressed with CA-rop1.
Supplemental Movie S5. FRAP analysis of NtPPME1-GFP at cell plate
formation in BY2 cells coexpressed with DN-rop1.
ACKNOWLEDGMENTS
We thank Zhenbiao Yang (University of Califonia, Riverside) for sharing the
rop1, DN-rop1, and CA-rop1 plasmids. Shengwang Du acknowledges the
support from NanoBioImaging Ltd. through a research contract with HKUST R
and D Corporation Ltd. (RDC14152140).
Received May 11, 2016; accepted August 8, 2016; published August 16, 2016.
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